Chromosome inheritance ensures transmission of genetic information. Abnormal chromosome segregation severely affects the health and viability of all cells and organisms. Errors in chromosome inheritance lead to chromosome abnormalities (aneuploidy) that cause many birth defects, such as Down Syndrome and miscarriages, and are the hallmarks of almost all types of cancers. The centromere, a complex of DNA and proteins, is central to the chromosome inheritance process and contributes to formation of the kinetochore, heterochromatin and sister chromatid cohesion. Defects in centromere functions lead to aneuploidy or tumor formation in yeast, flies and mice. CENP-A, a conserved H3-1ike histone that replaces H3 in centromeric nucleosomes, is the structural and functional foundation of the kinetochore and adjacent centromeric region. We have shown that kinetochore chromatin is uniquely arranged as interspersed domains of CENP-A and H3 nucleosomes that promote a specialized cylindrical structure on metaphase chromosomes. This special structure is formed on divergent DNA sequences, and it is not known how centromeric chromatin is established and maintained, nor the structural and functional aspects if it is altered. The goal of this proposal is to apply an integrative, dual-system approach, using novel reagents and techniques that I have developed in my former lab, to test key organizational and functional aspects of centromeric chromatin in Drosophila and humans. We propose to investigate the roles of genomic structure and chromatin organization and dynamics in assembly and regulation of CENP-A chromatin, centromeric domains, and metaphase kinetochore structure.